Life-stage and sex influence Philornis ectoparasitism in a Neotropical woodpecker (Melanerpes striatus) with essential male parental care

The nestlings of many Neotropical bird species suffer from Philornis (Diptera: Muscidae) ectoparasitism. While nestlings are typically considered the intended targets, recent work indicates that Philornis infest adult birds more frequently than previously appreciated, yet few studies have concurrently surveyed nestlings and adults for Philornis in the same population. Over six field seasons (2012 to 2017), I documented the presence of current or recent subcutaneous Philornis infestations on adult and nestling Hispaniolan Woodpeckers (Melanerpes striatus) from the same population. I tested the following three non-mutually exclusive hypotheses regarding occurrence of Philornis on adult birds: (1) nestlings are more vulnerable to Philornis parasitism than adults, (2) nesting is associated with Philornis parasitism in adults, and (3) Philornis parasitism is associated with incubation and brooding investment. While nestling and adult woodpeckers exhibited similar prevalence of parasitism, parasitized nestlings hosted on average 3.5 times more Philornis wounds (larvae plus scars) than parasitized adults. Nesting per se was not significantly associated with parasitism among adults, as breeding and non-breeding adults showed similar prevalence and intensity. However, adult males, which perform overnight incubation and brooding, were significantly more likely to be parasitized than adult females. This last result supports the hypothesis that incubation and brooding investment increase the risk of Philornis parasitism for adults, but this conclusion is complicated by the lack of an association between parasitism and nesting status. Together, these results raise questions about the degree of host life-stage specialization and whether adult parasitism is incidental or part of an alternative parasitic strategy for Philornis.

High ionic strength dissociation assay (HISDA) for high drug tolerant immunogenicity testing

Aim: Antidrug antibody (ADA) assessment may be challenged in studies that involve the administration of high doses of biotherapeutics and/or with long half-lives. In such cases, ADA assays with optimized drug tolerance are desired. Material & Methods: We evaluated the use of MgCl2 to develop high ionic strength dissociation assays in two investigational examples (bridging enzyme-linked immunosorbent ADA assays) to attain high drug tolerance while maintaining best possible structural integrity of ADAs. Results: Both ADA-bridging assays treated with MgCl2 showed improved drug tolerance and higher signal-to-blank values compared with overnight incubation or acid treatment. Conclusion: The use of MgCl2 treatment in ADA-bridging assays provides a sensitive, drug tolerant and easy-to-use alternative in cases where acid dissociation is not possible or unwanted.

High-throughput malaria serosurveillance using a one-step multiplex bead assay

Background Serological data indicating the presence and level of antibodies against infectious disease antigens provides indicators of exposure and transmission patterns in a population. Laboratory testing for large-scale serosurveys is often hindered by time-consuming immunoassays that employ multiple tandem steps. Some nations have recently begun using malaria serosurveillance data to make inferences about the malaria exposure in their populations, and serosurveys have grown increasingly larger as more accurate estimates are desired. Presented here is a novel approach of antibody detection using bead-based immunoassay that involves incubating all assay reagents concurrently overnight. Results A serosurvey in was performed in Haiti in early 2017 with both sera (n = 712) and dried blood spots (DBS, n = 796) collected for the same participants. The Luminex® multiplex bead-based assay (MBA) was used to detect total IgG against 8 malaria antigens: PfMSP1, PvMSP1, PmMSP1, PfCSP, PfAMA1, PfLSA1, PfGLURP-R0, PfHRP2. All sera and DBS samples were assayed by MBA using a standard immunoassay protocol with multiple steps, as well a protocol where sample and all reagents were incubated together overnight—termed here the OneStep assay. When compared to a standard multi-step assay, this OneStep assay amplified the assay signal for IgG detection for all 8 malaria antigens. The greatest increases in assay signal were seen at the low- and mid-range IgG titers and were indicative of an enhancement in the analyte detection, not simply an increase in the background signal of the assay. Seroprevalence estimates were generally similar for this sample Haitian population for all antigens regardless of serum or DBS sample type or assay protocol used. Conclusions When using the MBA for IgG detection, overnight incubation for the test sample and all assay reagents greatly minimized hands-on time for laboratory staff. Enhanced IgG signal was observed with the OneStep assay for all 8 malaria antigens employed in this study, and seroprevalence estimates for this sample population were similar regardless of assay protocol used. This overnight incubation protocol has the potential to be deployed for large-scale malaria serosurveys for the high-throughput and timely collection of antibody data, particularly for malaria seroprevalence estimates.

Rapid Rule Out of Culture-Negative Bloodstream Infections by Use of a Novel Approach to Universal Detection of Bacteria and Fungi

Background Currently it can take up to 5 days to rule out bloodstream infection. With the low yield of blood cultures (approximately 10%), a significant number of patients are potentially exposed to inappropriate therapy that can lead to adverse events. More rapid rule out can accelerate deescalation or cessation of antimicrobial therapy, improving patient outcomes. Methods A method is described, termed enzymatic template generation and amplification (ETGA), that universally and sensitively detects DNA polymerase activity liberated from viable bacteria and fungi isolated from blood culture samples as a measure of bloodstream infection. ETGA was applied in a diagnostic test format to identify negative blood cultures after an overnight incubation. Performance data for a prototype (Cognitor) and automated (Magnitor) version of the test are presented. Results The Cognitor manual assay displayed analytical reactivity for a panel of the 20 most prevalent causes of bloodstream infection, with a detection range of 28–9050 CFU/mL. Validation with 1457 clinical blood cultures showed a negative predictive value of 99.0% compared to blood culture incubation for 5 days. Magnitor showed an improved detection range of 1–67 CFU/mL, allowing for detection of bacteria-supplemented blood cultures after 2–8 h incubation, and Candida albicans-supplemented blood cultures at 16–22 h, 5–15 h faster than blood culture. Removing an aliquot from a blood culture bottle and replacing the bottle into the incubator was shown not to result in contaminating organisms being introduced. Conclusions The described method displays excellent breadth and detection for microbial cells and demonstrates the capability of confirming negative blood cultures after an overnight incubation in a blood culture instrument.

Maintenance of contractile function of isolated airway smooth muscle after cryopreservation

Isolated human airway smooth muscle (ASM) tissue contractility studies are essential for understanding the role of ASM in respiratory disease, but limited availability and cost render storage options necessary for optimal use. However, to our knowledge, no comprehensive study of cryopreservation protocols for isolated ASM has been performed to date. We tested several cryostorage protocols on equine trachealis ASM using different cryostorage media [1.8 M dimethyl sulfoxide and fetal bovine serum (FBS) or Krebs-Henseleit (KH)] and different degrees of dissection (with or without epithelium and connective tissues attached) before storage. We measured methacholine (MCh), histamine, and isoproterenol (Iso) dose-responses and electrical field stimulation (EFS) and MCh force-velocity curves. We confirmed our findings in human trachealis ASM stored undissected in FBS. Maximal stress response to MCh was decreased more in dissected than undissected equine tissues. EFS force was decreased in all equine but not in human cryostored tissues. Furthermore, in human cryostored tissues, EFS maximal shortening velocity was decreased, and Iso response was potentiated after cryostorage. Overnight incubation with 0.5 or 10% FBS did not recover contractility in the equine tissues but potentiated Iso response. Overnight incubation with 10% FBS in human tissues showed maximal stress recovery and maintenance of other contractile parameters. ASM tissues can be cryostored while maintaining most contractile function. We propose an optimal protocol for cryostorage of ASM as undissected tissues in FBS or KH solution followed by dissection of the ASM bundles and a 24-h incubation with 10% FBS before mechanics measurements.

Optimization of EDTA Exudation Technique for Proteome Study of the Phloem

Phloem proteome study is gaining recognition due to the phloem important function to a plant. Identification of proteins within the phloem with varying function could help understand the regulation of growth and development. EDTA exudation technique applied to obtain Arabidopsis phloem protein was optimized. It was found that plants grown up until 6 weeks in SD (short day) condition, exposed to 3 days LD (long day) condition, the phloem sap collected in 20 mM EDTA with overnight incubation in LD and precipitated using Amicon filter device gave the optimum amount of protein with good quality. Sufficient amount of protein was obtained using the optimized EDTA exudation technique for protein identification.

Detection and quantification of Xanthomonas axonopodis pv. phaseoli and its variant fuscans in common bean seeds

The common bacterial blight of common beans (CBCF), a disease caused by Xanthomonas axonopodis pv. phaseoli (Xap) and Xanthomonas axonopodis pv. phaseoli var. fuscans (Xapf), significantly reduces grain yield and seed quality. Because this bacterium is mainly disseminated through infected seeds, efficient detection of Xap and Xapf is important to assure the productivity and quality of the crop. In this study, various techniques that included different extraction techniques (two different incubation times, with and without centrifugation) and five culture media (Kado 523, GYCA, MXP, NSA, and PTSA) were tested for the detection of the seed-borne inoculum, using three different seed samples. Overnight incubation of the seeds, followed by centrifugation and incubation in Kado 523 resulted in higher extraction of Xap and Xapf. The best extraction technique was overnight incubation followed by centrifugation, and the best medium was PTSA. Among the tested culture media, PTSA provided better identification and counting of the bacterial colonies, thus allowing the quantification of the seed infection levels.

Decreased numbers of peripheral blood dendritic cells in patients with coronary artery disease are associated with diminished plasma Flt3 ligand levels and impaired plasmacytoid dendritic cell function

We investigated whether activation of circulating DCs (dendritic cells) or levels of Flt3L (FMS-like tyrosine kinase 3 ligand) and GM-CSF (granulocyte/macrophage colony-stimulating factor), haematopoietic growth factors important for DC differentiation, could account for reduced blood DC numbers in CAD (coronary artery disease) patients. Concentrations of Flt3L and GM-CSF were measured in plasma from CAD patients (n = 15) and controls (n = 12). Frequency and phenotype of mDCs (myeloid dendritic cells) and pDCs (plasmacytoid dendritic cells) were analysed by multicolour flow cytometry in fresh blood, and after overnight incubation with TLR (Toll-like receptor)-4 or -7 ligands LPS (lipopolysaccharide) or IQ (imiquimod). DC function was measured by IL (interleukin)-12 and IFN (interferon)-α secretion. Circulating numbers of CD11c+ mDCs and CD123+ pDCs and frequencies of CD86+ and CCR-7+ (CC chemokine receptor type 7) mDCs, but not pDCs, were declined in CAD. In addition, plasma Flt3L, but not GM-CSF, was lower in patients and positively correlated with blood DC counts. In response to LPS, mDCs up-regulated CD83 and CD86, but CCR-7 expression and IL-12 secretion remained unchanged, similarly in patients and controls. Conversely, pDCs from patients had lower CD83 and CCR-7 expression after overnight incubation and had a weaker IQ-induced up-regulation of CD83 and IFN-α secretion. In conclusion, our results suggest that reduced blood DC counts in CAD are, at least partly, due to impaired DC differentiation from bone marrow progenitors. Decreased levels of mDCs are presumably also explained by activation and subsequent migration to atherosclerotic plaques or lymph nodes. Although mDCs are functioning normally, pDCs from patients appeared to be both numerically and functionally impaired.

The Immunoregulatory Enzyme Indoleamine 2,3-Dioxygenase (IDO1) Is Expressed by Natural Killer (NK) Cells During Cytokine-Mediated Activation

Abstract 3894 Introduction: human NK cells are large, granular cells derived from lymphocyte lineage. They are critical mediators of the innate immunity as they rapidly respond to pathogens infected and tumour cells through cytotoxic and cytokines-producing responses. Indoleamine 2,3-dioxygenase (IDO1) is an enzyme catalyzing the degradation of the essential amino acid L-tryptophan into kynurenines. Several cell types, including dendritic cells, have been shown to express IDO1, which acts as a potent immunosuppressive agent. Recently, in addiction to IDO1, the new variant IDO2 was described but its immunosuppressive role is still under investigations. The aim of the present work is to study the expression and the role of IDO1 and IDO2 in NK cells. Methods: CD3-CD56+NK cells were immunomagnetically purified from healthy donors. IFN-gamma production was measured by ELISA assay. IDO1 and DAP12 transcript levels were normalized on ABL. IDO2 expression was measured by semi-quantitative PCR. Kynurenine levels were evaluated by colorimetric assay. Results: peripheral blood NK cells were treated with IL-2 for 16h, 40h, 88h and 160h. At the end of each IL-2 stimulation, supernatants were collected and IFN-gamma production was measured by colorimetric assay. IDO1 expression in NK cells was evaluated by quantitative real-time PCR and Western blotting. Our data show that, during IL-2-mediated activation, IDO1 is up-regulated at mRNA and protein levels. This effect is maximum after an overnight incubation and it decreases at later time points. IDO1 expression is clearly correlated with IFN-gamma production by NK cells whereas IFN-gamma receptor expression is not affected. Culturing NK cells with a combination of IL-2 and anti-IFN-gamma antibody demonstrated that IDO1 expression is regulated, at least in part, by IFN-gamma. Interestingly, IDO1 mRNA upregulation is associated with downregulation of DAP12, a gene whose expression has been showed to be inversely correlated to IDO1 in dendritic cells. IDO2 expression seems to be not affected by IL-2 stimulation. Treatment with other inflammatory cytokines demonstrated that IDO1 is modulated by different stimuli. Overnight incubation with IL-12, IFN-gamma and a combination of IL-2 and IL-6 showed IDO1 induction. IFN-gamma showed the highest IDO1 induction at transcript and protein level and, surprisingly, of IDO2 expression. IDO1 expression by NK cells resulted in increased production of kynurenines and this effect was abrogated by the addition of the IDO1 inhibitor 1-methyl tryptophan. Conclusion: our results demonstrate that NK cells upregulate IDO1 expression during cytokine-mediated activation and that IDO1 expression is regulated, at least in part, by IFN-gamma. Similarly to DCs, NK cells express IDO1 gene in association with DAP12, thus suggesting a common regulatory pathway among different cell subsets. Disclosures: Baccarani: NOVARTIS: Honoraria; BRISTOL MYERS SQUIBB: Honoraria.